Air-sampled Filter Analysis for Endotoxins and DNA Content

Summary

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Abstract

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Introduction

Air sampling on filters is a basic tool in atmospheric aerosols research.¹ The sampled filters are the starting point for various chemical, physical, and biological characterizations of the collected ambient particles.^2-11 The advantage of this approach is that various analyses can be performed off-line on the same sample. Compiling the data from all the different analyses enables the researcher to obtain a good understanding of the characteristics of the collected particles and aids in solving complex questions in the atmospheric sciences.^12,13 For example, marine and inland air-samples taken during the same period can be compared wit....

Protocol

Note: A detailed list of all the materials and instruments used in this protocol is shown in the Materials section.

1. Air Sampling on Filters

1. Preparation of Filters
   1. For high volume sampling, use 20.3 x 25.4 cm² filters. Choose the specific type of filter that best fits the research needs, as well as the filter cut-off size, if applicable.¹ Here, use quartz microfiber filters.
   2. Pre-bake filters intended for organic and biological compound sampling to destroy organic residues. To pre-bake the filters, wrap them individually in aluminum foil and then bake them in a laboratory furnace at ....

Results

It is common to study atmospheric aerosols using "off-line" analyses of sampled filters (see Figure 2).³² Chemical analyses of the sampled matter include organic (e.g. protein, hydrocarbon molecules, saccharides) and inorganic (e.g. metals, salts) content. Biological analyses include viable and non-viable microorganism content, species identification using a DNA approach or microscopy, as well as DNA-based quantification.

Discussion

This work describes extraction and detection methods for quantifying both endotoxins and DNA present in aerosol samples collected on filters. The methods require accurate routines and can be performed easily as long as the experimentalist adheres to a few essential and important points discussed here.

For the endotoxin detection step, note that the lysate solution is quite viscous and tends to produce bubbles upon pipetting. It is difficult to remove thee bubbles, and they lead to changes in t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Filter sampling
HiVol 3000 - High Volume Air Sampler	Ecotech
Quartz Microfiber Filters	Whatman	1851-865	203 mm x 254 mm
ELF - Laboratory Chamber Furnaces	Carbolite	ELF 11series
Aluminum foil	Opal
Name	Company	Catalog Number	Comments
Endotoxin
Ethanol	Sigma Aldrich	16368	Laboratory Reagent, 96%
Airstream Class II Biological Safety Cabinet AC-4E1	ESCO	10011712
Pyrotell -T	Associates of Cape Cod, Inc.	T0051
Control Standard Endotoxin	Associates of Cape Cod, Inc.	E0005-1	Escherichia coli O113:H10, 0.5 µg/vial 1 Pack
LAL Reagent Water	Associates of Cape Cod, Inc.	W0051
10 ml sterile syringe with Luer-Lok Tip	Becton-Dickinson & Co.	309605
BD Precisionglide syringe needle	Becton-Dickinson & Co.	305129	Sterile
Parafilm-M sealing tape	Parafilm	P7543	Sigma catalog number
Microtubes	Axigen	MCT-200-C	2 ml, pyrogen free
1.12 cm diameter Cork Borer	Boekel Scientific	1601 BD Series - Steel	Part of a cork borer set containing borers with various diameters.
50 mm Petri Dish	Miniplast Ein-Shemer	72050-01	Aseptic
Vortex Genie 2	Scientific Industries, inc.	SI-0297
Microcentrifuge 5415 D	Eppendorf	22621408
TC MicroWell 96 F SI w/lid	Nunc	167008	Flat bottom wells (with lid (individually wrapped)), sterile, pyrogen free
Synergy HT Multi-Detection Microplate Reader	Biotek	7091000
Name	Company	Catalog Number	Comments
DNA
DNA away	Sigma Aldrich	7010
Standard DNA of the microbial species of interest	ATCC or other culture collection		Either the appropriate microbial strain for DNA extraction or the extracted DNA
Neubauer-improved	Marienfeld	640030	hemocytometer
TE buffer, Low EDTA	Life Technologies	12090-015	10 mM Tris-HCl (pH 8.0) 0.1 mM EDTA
Nuclease-free PCR-grade water	Sigma Aldrich	3315959001
PCR primers	Sigma Aldrich		Targets the microbial species of interest
Dual-Labeled Probes	Sigma Aldrich		Targets the microbial species of interest
Screw cap tubes	Axigen	ST-200-SS	2 ml
PowerSoil DNA extraction kit	Mo Bio Laboratories	12888-100
Glass beads, acid-washed 425-600 µm	Sigma Aldrich	G8772-100G
Glass beads, acid-washed <106 µm	Sigma Aldrich	G4649-100G
PowerSoil Solution C1	Mo Bio Laboratories	12888-100-1	Cell lysis buffer , Power soil Kit
Magic Touch ice bucket	Bel-Art	18848-4001
Mini-Beadbeater-16	BioSpec	607EUR
StepOnePlus Real-Time PCR System	Applied Biosystems	4376600
Fast SYBR Green Master Mix	Applied Biosystems	4385612
TaqMan Gene Expression Master Mix	Applied Biosystems	4370048
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 ml	Applied Biosystems	4346906
MicroAmp Splash-Free 96-Well Base	Applied Biosystems	4312063
MicroAmp Optical Adhesive Film	Applied Biosystems	4311971
Centrifuge 5810 R	Eppendorf	5811 000.010	Rotor A-4-62 with MTP buckets